Abstract
CDAP (1-cyano-4-dimethylaminopyridine tetrafluoroborate) is employed in the synthesis of conjugate vaccines as a cyanylating reagent. In the published method, which used pH 9 activation at 20 °C (Vaccine, 14:190, 1996), the rapid reaction made the process difficult to control. Here, we describe optimizing CDAP activation using dextran as a model polysaccharide. CDAP stability and reactivity were determined as a function of time, pH and temperature. While the rate of dextran activation was slower at lower pH and temperature, it was balanced by the increased stability of CDAP, which left more reagent available for reaction. Whereas maximal activation took less than 2.5 min at pH 9 and 20 °C, it took 10–15 min at 0 °C. At pH 7 and 0 °C, the optimal time increased to >3 h to achieve a high level of activation. Many buffers interfered with CDAP activation, but DMAP could be used to preadjust the pH of polysaccharide solutions so that the pH only needed to be maintained. We found that the stability of the activated dextran was relatively independent of pH over the range of pH 1–9, with the level of activation decreased by 40–60% over 2 h. The use of low temperature and a less basic pH, with an optimum reaction time, requires less CDAP, improving activation levels while making the process more reliable and easier to scale up.
Highlights
The development of conjugate vaccines is one of the great advances in vaccinology
We have found that the aqueous CDAP solution was reasonably stable as the extent of protein conjugation to CDAP-activated dextran was comparable, whether the pH was raised at 30 s or 5 min after CDAP addition
Having identified that CDAP rapidly hydrolyzes at pH 9 at room temperature, the standard activation conditions, we investigated the hypothesis of slowing down CDAP hydrolysis by reducing the temperature
Summary
The development of conjugate vaccines is one of the great advances in vaccinology. As T-cell independent antigens, polysaccharides are poorly immunogenic in infants [1]. These shortcomings have been overcome by chemically linking a polysaccharide to a carrier protein to form a conjugate vaccine, as first observed by Goebel and Avery [2,3]. Conjugate vaccines for Streptococcus pneumoniae and meningococcal disease are routinely administered. These vaccines, which are often multivalent, are complex and challenging to manufacture [5]
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